The present invention relates to a substantially endotoxin-free apolipoprotein A (ApoA) or apolipoprotein E (ApoE) and a process for producing the same, by separating the endotoxins from the ApoA or ApoE, or variants or mixtures thereof, by contacting a first aqueous solution containing said ApoA or ApoE with a matrix containing an immobilized compound with an end group comprising two or three nitrogen atoms bonded to a carbon atom, and subsequently treating the matrix containing an immobilized compound with a second aqueous solution containing a surfactant, or by contacting a first aqueous solution containing said ApoA or ApoE with an anion-exchange matrix, and subsequently treating the anion-exchange matrix with a second aqueous solution containing a compound comprising two or three nitrogen atoms bonded to a carbon atom. The invention further relates to use of a matrix containing an immobilized compound comprising two or three nitrogen atoms bonded to a carbon atom and a solution containing a surfactant, or an anion-exchange matrix and a solution containing a compound comprising two or three nitrogen atoms bonded to a carbon atom, for removing endotoxins from aqueous solutions containing ApoA or ApoE, or variants or mixtures thereof. The thus produced ApoA or ApoE can be used for the manufacture of a medicament in the treatment of atherosclerosis and cardiovascular diseases, as well as in a method for treatment of atherosclerosis and cardiovascular diseases when administered in a therapeutically effective amount.
The clear correlation between elevated levels of serum cholesterol and the development of coronary heart disease (CHD) has been repeatedly confirmed, based on epidemiological and longitudinal studies. The definition, however, of complex mechanisms of cholesterol transport in plasma, has allowed the recognition of a selective function of circulating lipoproteins in determining the risk for CHD.
There are, in fact, four major circulating lipoproteins: chylomicrons (CM), very low density (VLDL), low density (LDL) and high density (HDL) lipoproteins. Of these, HDL is directly involved in the removal of cholesterol from peripheral tissues, carrying it back either to the liver or to other lipoproteins, by a mechanism known as xe2x80x9creverse cholesterol transportxe2x80x9d (RCT).
The xe2x80x9cprotectivexe2x80x9d role of HDL has been confirmed in a number of studies. Recent studies directed to the protective mechanism(s) of HDL have been focused on apolipoprotein A-I (ApoA-I), the major component of HDL. High plasma levels of ApoA-I are associated with a reduced risk of CHD and presence of coronary lesions.
Plasma ApoA-I is a single polypeptide chain of 243 amino acids, whose primary sequence is known (Brewer et al. (1978) Biochem. Biophys. Res. Commun. 80: 623-630). ApoA-I is synthesized as a 267 amino acid precursor in the cell. The major structural requirement of the ApoA-I molecule is believed to be the presence of repeat units of 11 or 22 amino acids, presumed to exist in amphipathic helical conformation (Segrest et al. FEBS Lett. (1974) 38: 247-253). This, structure allows for the main biological activities of ApoA-I, i.e. lipid binding and lecithin cholesterol acyl transferase (LCAT) activation.
The apolipoprotein A-IMilano (ApoA-IM) is the first described molecular variant of human ApoA-I (Franceschini et al. (1980) J. Clin. Invest. 66: 892-900). It is characterized by the substitution of Arg 173 with Cys 173 (Weisgraber et al. (1983) J. Biol. Chem. 258:2508-2513). The mutant apolipoprotein is transmitted as an autosomal dominant trait and 8 generations of carriers have been identified (Gualandri et al. (1984) Am. J. Hum. Genet. 37: 1083-1097). The status of a ApoA-IM carrier individual is characterized by a remarkable reduction in HDL-cholesterol level. In spite of this, the affected subjects do not apparently show any increased risk of arterial disease. Indeed, by examination of the genealogical tree it appears that these subjects may be xe2x80x9cprotectedxe2x80x9d from atherosclerosis.
The mechanism of the possible protective effect of ApoA-IM in the carriers seems to be linked to a modification in the structure of the mutant apolipoprotein, with the loss of one alpha-helix and an increased exposure of hydrophobic residues (Francheschini et al. (1985) J. Biol. Chem. 260: 1632-1635). The loss of the tight structure of the multiple alpha-helices leads to an increased flexibility of the molecule, which associates more readily with lipids, compared to normal ApoA-I.
Another very specific feature of the ApoA-IM, is its capacity to form dimers with itself and complexes with ApoA-II, in both cases because of the presence of the Cys residue.
To make possible production of sufficient quantities of ApoA-I in general, and more specifically ApoA-IM, use is made of recombinant DNA techniques, e.g. in E. coli. Thus, recombinant preparation and use of ApoA-IM, monomers as well as dimers, are disclosed in patent specifications WO-A-88/03166 assigned to Farmitalia Carlo Erba (FICE), WO-A-90/12879 assigned to Sirtori et al, as well as WO-A-93/12143 and WO-A-94/13819 both assigned to Pharmacia AB (formerly Kabi Pharmacia AB).
Use of e.g. E. coli as medium introduces certain drawbacks. Thus, endotoxins or lipopolysaccharides (LPS) are high molecular complexes associated with the outer membrane (cell wall) of grain-negative bacteria, such as E. coli, Proteus and Salmonella. Endotoxins consist of two main parts, a lipid moiety called lipid A which is embedded in the outer membrane and a polysaccharide (O-antigen) which protrudes into the environment. Lipid A is the region which elicit the toxic effect of the endotoxins, a prerequisite being the presence of the entire lipid A moiety. The polysaccharide is made up of a O-specific chain and a core. The O-specific chain projects from the core and is the outermost part of the endotoxin. The core works as a linkage between lipid A and the O-specific chain.
It is known that endotoxins must be released from the bacterial surface to cause toxic effects. This happens when the bacteria multiply, at lysis and during stress. In aqueous solutions, free endotoxins form aggregates, micelles and vesicles, with a molecular weight of about 5 kDa up to greater than 103 kDa.
It is known from the literature that several proteins form complexes with endotoxins. Particularly strong complexes are formed with HDL and apolipoproteins (Emancipator et al. (1992) Infect. Immun. 60: 596-601). According to Ulevitch et al. (1981) J. Clin. Invest. 67: 827-837, formation of a complex between HDL and endotoxins involve a two step mechanism, as follows:
Endotoxins(aggregated)xe2x86x92Endotoxins(disaggregated)xe2x80x83xe2x80x83(1)
Endotoxins(disaggregated)+HDLxe2x86x92Endotoxins-HDLxe2x80x83xe2x80x83(2)
This behavior has been confirmed e.g. by Munford et al (1981) Infect. Immun. 34: 835-843. There are indications suggesting that lipid A is the main factor in the complex and that the interaction involves both ionic and hydrophobic forces (Freudenberg et al. (1979) Nat. Toxins, Proc. Int. Symp. Anim., Plant Microb. Toxins., 6th, 349-354).
As already stated above, strong complexes are formed between endotoxins and HDL in general and particularly with apolipoproteins. This mechanism has been used in U.S. Pat. No. 5,128,318 assigned to the Rogosin Institute. U.S. Pat. No. 5,128,318 thus relates to HDL associated apolipoprotein containing reconstituted particles, and use thereof in removing lipid soluble materials, including endotoxins, from cells, body fluids, and the like. More particularly, U.S. Pat. No. 5,128,318 relates to a method for treating a subject for endotoxin-caused toxicity, by administering to the subject a reconstituted particle containing ApoA-I or ApoA-II, with or without cholesterol. Here, naturally, the aim is to create and maintain indefinitely the strongest possible complex, to avoid release of the endotoxins in the subject.
The complexes, strong in themselves, can be further strengthened e.g. by the presence of certain chemical compounds. Thus, deoxycholate is known to disaggregate endotoxins according to formula (1) above (Munford et al., see above and Emancipator et al., see above). The deoxycholate, then increases the binding of endotoxins to HDL according to formula (2). The result is a complex of endotoxins and HDL, which is very difficult to separate.
General methods for reducing or eliminating the effect of endotoxins are known previously. Thus, EP-A-494848 assigned to Pharmacia discloses methods for inhibiting endotoxin induced effects. A first embodiment relates to infusing a medicament containing arginine or arginine derivatives for treatment of an endotoxin induced effect, e.g. fever. A second embodiment relates to a method for removing endotoxins from water or aqueous solutions by filtering the water or aqueous solution through a bed containing immobilized arginine or an arginine derivative. To illustrate the second embodiment, tests were carried out with endotoxins from E. coli on columns containing Arginine Sepharose(copyright) from Pharmacia Biotech of Uppsala, Sweden. The interaction is, however, weak and therefore much easier to separate into protein and endotoxins than would be the case with the strong complexes between apolipoproteins and endotoxins.
Anion-exchange chromatography is frequently used in the elimination of endotoxins from solutions containing proteins such as urokinase, interferon, asparaginase and albumin (Sharma (1986) Biotech. Applied Biochem. 8:5-22). However, the interaction between the proteins and endotoxins is much weaker than the complexes formed between apolipoproteins and endotoxins.
EP-A-333474 to Mitsui Toatsu relates to a process for removing endotoxins from proteins by contacting an endotoxin-contaminated aqueous solution containing the protein with a protein adsorbent, washing the adsorbent with a solution containing an amino compound, and subsequently eluting the protein from the adsorbent. The only exemplified proteins are tissue plasminogen activator (t-PA), human serum albumin and inter-xcex1-trypsin inhibitor. Examples of protein adsorbents are affinity, adsorption, hydrophobic and metal chelate chromatography gels.
Polymyxin B sulfate is an antibiotic polypeptide which has the ability to prevent the toxic effects of endotoxin by interaction with the lipid A moiety. Karplus and coworkers (Karplus et al. (1987) J. Immuno. Methods 105: 211-220) have used this knowledge for adsorbing endotoxins on Polymyxin Sepharose(copyright) 4B, sold by Pharmacia AB of Uppsala, Sweden. Polymyxin is, however, in itself biologically active and therefore not suitable for removing endotoxin from solutions for intravenous injection (H. Matsumae et al. (1990) Biotechn. Biochem. 12: 129-140).
Prosep(copyright) Remtox sold by Bioprocessing Ltd. of Great Britain, is a matrix prepared for specific removal of endotoxins from low and high molecular weight substances, such as antibiotics, vitamins, enzymes, antibodies and blood products. The gel consists of a low molecular weight, non-protein, non-carbohydrate synthetic ligand.
Charged filters are capable of removing endotoxins and other negatively charged molecules from different solutions. For example, Pall of United Kingdom offers Posidyne(trademark) filters, consisting of a hydrophilic nylon 66 filter medium containing quaternary ammonium groups throughout the membrane structure. The retention capacity of the filter is independent of the temperature and is optimal at a pH of 5-8 and at a low flow rate.
There are presently several methods known for reducing or eliminating the influence of endotoxins in protein solutions generally. There is, however, no existing method to overcome the strong interaction between endotoxins and ApoA or ApoE. The present invention is intended to solve this problem.
An object of the present invention is to provide an efficient purifying process, for producing ApoA or ApoE with a very low content of endotoxins.
Another object of the present invention is to provide an efficient process, where the activity of the ApoA or ApoE is essentially retained.
A further object of the present invention is a process providing a high yield of ApoA or ApoE, i.e. a process with a minimal loss of product.
The objects above are met by the present invention, which relates to a process for separating endotoxins from apolipoprotein A (ApoA) or apolipoprotein E (ApoE), or variants or mixtures thereof, by contacting a first aqueous solution containing said ApoA or ApoE with a matrix containing an immobilized compound with an end group comprising two or three nitrogen atoms bonded to a carbon atom, and subsequently treating the matrix containing an immobilized compound with a second aqueous solution containing a surfactant, or by contacting a first aqueous solution containing said ApoA or ApoE with an anion-exchange matrix, and subsequently treating the anion-exchange matrix with a second aqueous solution containing a compound comprising two or three nitrogen atoms bonded to a carbon atom.
The inventors of the present invention have surprisingly found that matrices containing e.g. immobilized arginine, guanidine or histidine, can be used to strongly attach the endotoxins, and thereby the ApoA or ApoE, to the matrix. By subsequently eluting with a surfactant-containing solution, the ApoA or ApoE molecules can be released, while the endotoxins remain attached to the matrix. It is also possible to strongly attach the ApoA or ApoE and thereby the endotoxins to an anion-exchange matrix. By subsequently eluting with a solution containing e.g. urea or arginine, or salts of guanidine or histidine, the endotoxins can be released, while the ApoA or ApoE molecules remain attached to the matrix. Finally, the ApoA or ApoE molecules can be released from the matrix by increasing the ionic strength.
The selection of conditions under which to perform the present process will be governed by a desire to reach as low concentration of the endotoxins as possible, while at the same time obtain an acceptable recovery of the ApoA or ApoE.
With the present process it is possible to produce ApoA or ApoE which are substantially endotoxin-free. In the present invention, substantially endotoxin-free means a concentration below about 1 EU/mg of ApoA or ApoE. Specifically, it is possible to produce substantially endotoxin-free ApoA or ApoE which have been produced by recombinant DNA technique, more specifically in gram-negative bacteria, and even more specifically in E. coli. 
With the present process it is possible to produce ApoA or ApoE with a low content of endotoxin in combination with a protein recovery of at least 70%, suitably at least 80%, preferably at least 90% and more preferably at least 95%.
The present invention also relates to use of a matrix containing an immobilized compound with an end group comprising two or three nitrogen atoms bonded to a carbon atom and a solution containing a surfactant, or an anion-exchange matrix and a solution containing a compound comprising two or three nitrogen atoms bonded to a carbon atom, for removing endotoxins from aqueous solutions containing ApoA or ApoE, or variants or mixtures thereof.
The present invention further relates to use of ApoA or ApoE produced according to the inventive process for the manufacture of a medicament comprising the ApoA or ApoE in the treatment of atherosclerosis and cardiovascular diseases.
The present invention further relates to a method for treatment of atherosclerosis and cardiovascular diseases, by administering ApoA or ApoE produced according to the inventive process in a therapeutically effective amount.
In a first embodiment, an aqueous solution containing ApoA or ApoE is loaded onto a matrix with immobilized ligands with an end group comprising two or three nitrogen atoms bonded to a carbon atom. Subsequently, the complex of ApoA or ApoE and endotoxins is separated by eluting with an aqueous solution containing a surfactant, whereby the ApoA or ApoE is released while the endotoxins remain attached to the ligands. Finally, the matrix is regenerated by washing with one or more liquids containing various combinations of e.g. NaOH, C2H5OH, HAc and NaAc.
Examples of end groups that can be used in the present invention are those containing a guanidyl group, e.g. arginine and guanidine, or a heterocyclic group, e.g. histidine. The end groups are suitably non-heterocyclic, preferably containing a guanidyl group rendering the end groups strong bases. The end group containing a guanidyl group is more preferably arginine or guanidine, most preferably arginine. The end groups can be bonded directly to the matrix. More commonly, however, the end group is bonded to the matrix through a spacer, which can be inert or exhibit additional binding capacity. Spacers well suited for the present process can be found e.g. in Arginine bonded to Sepharose(copyright) and Histidine bonded to Minileak(copyright).
In a second embodiment, an aqueous solution containing ApoA or ApoE is loaded onto an anion-exchange matrix. Subsequently, the complexes of ApoA or ApoE and endotoxins are separated by eluting with an aqueous solution containing a compound comprising two or three nitrogen atoms bonded to a carbon atom. In this way the endotoxins are released while the ApoA or ApoE remain attached to the ligands. Suitable examples of the nitrogen-containing compounds are urea, arginine and guanidine hydrochloride. Finally, the ApoA or ApoE are released from the matrix by increasing the ionic strength, suitably to about 0.5 up to about 2 M, preferably by addition of NaCl.
Conventionally, in processes for purifying proteins the concentrations of e.g. urea and guanidine hydrochloride are kept at a minimum to avoid irreversible denaturation of the protein at issue. Surprisingly, the inventors have found that a higher than conventional concentration of the nitrogen-containing compound is advantageous when carrying out the present invention, since it facilitates the uncovering of the strong complex between endotoxins and ApoA. Thus, the concentration of urea should be in the range of from about 0.75 M up to saturation at the prevailing temperature, suitably in the range of from 2.5 M up to 8 M, preferably from 4.5 up to 7.5 M. It lies within the competence of the person skilled in the art to select the corresponding concentrations for e.g. guanidine hydrochloride and arginine.
In a third embodiment, the first and second embodiments are combined so as to give a product with a very low level of endotoxin. Thus, the aqueous solution containing ApoA or ApoE can be loaded onto an anion-exchange step whereafter the endotoxins are released by eluting with a compound comprising two or three nitrogen atoms bonded to a carbon atom. The ApoA or ApoE with a reduced concentration of endotoxins, are released from the anion-exchange matrix, and, optionally after one or more intermediate process steps such as a desalting step, the resulting ApoA or ApoE is loaded onto a matrix with immobilized compounds with end groups comprising two or three nitrogen atoms bonded to a carbon atom. Finally, the ApoA or ApoE can be released in a very pure form. The reversed sequence, i.e. a first step with a matrix containing immobilized ligands, and a second step with an anion-exchange matrix, can also be used to advantage in the present invention.
The matrices of the present invention can be soluble or insoluble in various common solvents, e.g. organic polymers soluble or insoluble in water with or without ethanol. Matrices also include e.g. filters to which ligands comprising two or three nitrogen atoms bonded to a carbon atom have been coupled.
The immobilized compounds with an end group comprising two or three nitrogen atoms bonded to a carbon atom can be supported on any inorganic or organic matrix. Thus, the matrix can be selected from various strongly hydrophilic matrices e.g. agarose matrices such as a wide variety of Sepharose(copyright) matrices sold by Pharmacia Biotech of Uppsala, Sweden, organic polymer matrices such as TSK-gels sold by Tosoh Corp. of Tokyo, Japan, or highly porous organic polymer matrices sold by Per Septive Bio-systems of Boston, USA. The matrix is preferably an agarose matrix. Suitable agarose matrices in the present invention are, apart from Sepharose(copyright), Minileak(copyright) sold by Kem-En-Tec A/S of Copenhagen, Denmark and Bio-Gel A sold by Bio-Rad, of Brussels, Belgium. Preferably, the matrix is cross-linked allowing for a fast flow (FF) and thereby high production capacity.
Anion-exchange matrices useful in a process according to the second embodiment, are e.g. agarose matrices such as DEAE Sepharose(copyright) and Q Sepharose(copyright) matrices sold by Pharmacia Biotech of Uppsala, Sweden. Further examples of anion-exchange matrices that can be used in the present process are Super Q-650 and Fractogel EMD DEAE-650 sold by Toso Haas of Tokyo, Japan, and Hyper D sold by Biosepra S.A. of France. The anion-exchange matrix is suitably an agarose matrix. Preferably, the anion-exchange matrix is cross-linked allowing for a fast flow (FF) and thereby high production capacity.
The present invention is advantageously used for removing endotoxins from any apolipoprotein A (ApoA) or Apolipoprotein E (ApoE), or variants or mixtures thereof. The present invention is especially suitable when the ApoA or ApoE are produced by a recombinant DNA technique in gram-negative bacteria, and in particular when they are produced in E. coli. In the present invention, the terms ApoA and ApoE include any preform or fragment, or any truncated, extended or mutated form, or any mixture of any of these forms or fragments. Preform relates e.g. to the 249 amino acid Met form of ApoA-I as disclosed in WO-A-88/03166 assigned to Sirtori et al. Other preforms are the proapolipoprotein A-Is disclosed in U.S. Pat. No. 5,059,528 to UCB as well as EP-A-308336, JP 216988/1984 and JP 252048/1987 all to Mitsubishi Chem. Ind. Fragment relates to a part of ApoA or ApoE containing at least one alpha helix, e.g. as disclosed in WO-A-93/25581 assigned to Innogenetics S.A. of Belgium. Truncated and extended forms relate to ApoA and ApoE molecules where one or more amino acid is missing or has been added, respectively, at the N and/or C terminal ends of the molecules. Suitably, from two up to eight amino acids are missing or have been added, preferably from three up to six amino acids. Mutated forms relate to ApoA and ApoE molecules where one or more amino acid has been substituted by another amino acid, e.g. ApoA-IM as disclosed in WO-A-93/12143 and WO-A-94/13819. Other mutated forms are ApoA-ISeattle (Deeb et al (1991) J. Bio. Chem. 266:13654-13660), ApoA-IYame (Takada et al (1991) J. Lipid Res. 32: 1275 ff) and a yet unnamed mutated form of ApoA-I (Matsunaga et al (1991) Proc Natl. Acad. Sci. USA 88:2793-2797).
Human ApoE and variants thereof, are disclosed in xe2x80x9cHuman Apolipoprotein Mutants IIIxe2x80x9d, ed. by C. R. Sirtori et al (1993) Nato ASI Series, Springer Verlag, Berlin, II 73:81-96.
The present invention can be used to advantage for removing endotoxins from ApoA as well as ApoE. In the following description, however, use will be made of ApoA to further describe the present invention.
Known ApoAs are e.g. ApoA-I, ApoA-II and ApoA-IV. In the present invention, suitably, the ApoA is ApoA-I, or variants or mixtures thereof. Natural ApoA-I is a single polypeptide chain, composed of 243 amino acids. More suitably, the ApoA is a mutated form of ApoA-I where at least one Cys residue has been substituted for a Arg residue making formation of disulfide-hinked dimer possible. In the amino acid sequence of natural human ApoA-I, Arg residues are located at positions 10, 27, 61, 83, 116, 123, 131, 149, 151, 153, 160, 171, 173, 177, 188 and 215. Of these, substitutions are preferred at one or more of positions 160, 171, 173, 177 and 188, i.e. at positions within the same alpha helix. More preferably, the Arg residue is substituted at positions 171 and/or 173.
Human apolipoprotein A-IMilano (ApoA-IM) is a naturally occurring mutated form of normal ApoA-I (Weisgraber et al. (1980) J. Clin. Invest. 66: 901-907). In ApoA-IM, one residue of the amino acid arginine (Arg 173) has been replaced by a residue of the amino acid cysteine (Cys 173). Since ApoA-IM contains one cysteine residue per polypeptide chain, it may exist as a monomer or as a disulfide-linked dimer. The molecular weight of the monomer is about 28,000 Da and for the dimer about 56,000 Da. These two forms are chemically interchangeable; and the term ApoA-IM does not, in the present context, discriminate between the two forms.
The initial concentration of endotoxin in the aqueous solutions containing ApoA, may be more than 106, more than 107, and even more than 108 EU/mg of protein. The final concentration of endotoxin can be reduced to below about 100 EU/mg, by applying the present invention to the aqueous solutions containing ApoA. Suitably, the final concentration of endotoxin is reduced to below about 10 EU/mg, and preferably to below about 1 EU/mg, rendering the Apo A substantially endotoxin-free.
To reach low levels of endotoxin in the aqueous solutions containing ApoA, it may be necessary to recirculate the solution so that it is contacted with the matrix at least twice. It is of course also possible to use the same or different matrices in at least two consecutive steps, to reach a sufficiently low level of endotoxins. In any of these ways, it is possible to reduce, the initial concentration of endotoxins by at least 104, suitably by at least 105, and preferably by at least 106 in the process.
The matrix is normally equilibrated with a first buffer before the sample containing ApoA is loaded onto the matrix. After loading, the matrix is treated with a second buffer to elute the essentially endotoxin-free ApoA from the matrix.
In a preferred embodiment, the first aqueous solution containing ApoA further contains a surfactant. The surfactant is added in order to at least partially disaggregate the complexes between the endotoxins and ApoA before contacting said first aqueous solution with the matrix. Thus, by maintaining the first aqueous solution containing a surfactant for at least 5 min before contacting said first aqueous solution with the matrix, the interaction between the complex and the particular ligand is facilitated. Suitably, the surfactant is added and the resulting solution maintained for a period of time in the range of from 15 min up to 10 h, preferably from 30 min up to 4 h, before being contacted with the matrix.
It is a prerequisite in the first embodiment and preferred in the second embodiment, that the elution buffer contains a surfactant, suitably an anionic one such as sodium dodecyl sulfate (SDS). The surfactant enhances the effect of the compound comprising two or three nitrogen atoms bonded to a carbon atom, probably by uncovering the strong complex between endotoxins and ApoA, and subsequently separating the endotoxins from ApoA. The separation is most probably controlled by reaction kinetics, for which reason the period of time before equilibrium is reached can be substantial.
Examples of surfactants which can be used to advantage in the present invention are various bile acids or salts thereof, such as sodium deoxycholate and sodium cholate. Also, non-ionic surfactants, e.g. zero-net-charge surfactants such as polyoxyethylene sorbitan fatty esters, block co-polymers and alkyl ethoxylates, can be used to advantage in the present invention. Examples of polyoxyethylene sorbitan tan fatty esters are polyoxy-ethylene-(20)-sorbitan monolaurate, e.g. Tween(copyright) 80, and polyoxy-ethylene-(20)-sorbitan monooleate, e.g. Tween(copyright) 20, both sold by ICI of Great Britain. Examples of the block co-polymers are combinations of polypropyleneglycol and polyethyleneglycol, e.g. Pluronic(copyright) sold by BASF in Germany. Examples of alkyl ethoxylates are Triton(copyright) X-100 and Triton(copyright) X-114 sold by Union Carbide in USA.
In the present invention, surfactant also includes various lipids, which can be natural or synthetic compounds consisting of acyl carriers, such as glycerides, sphingosine, cholesterol, or derivatives or mixtures thereof, to which one or more fatty acids can be bonded. The lipids can, depending on their polarity, be divided into non-polar, polar and amphiphilic lipids. Examples of non-polar lipids are monoacylglycerides, diacylglycerides, triacylglycerides, and cholesterol. Examples of polar and amphiphilic lipids are phospholipids and glycolipids. Suitably, the polar and amphiphilic lipids are bilayer forming, such as phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidylglycerol, phosphatidyletanolamine, phosphatidylserine, sphingomyelin, or mixtures thereof. The natural lipids can be produced from e.g. soybean oil, maize oil, soy lecithin and egg lecithin. Other suitable examples are synthetic and saturated or unsaturated PC:s, such as dipalmitoyl phosphatidylcholine (DPPC) and dimyristyl phosphatidylcholine (DMPC).
The concentration of surfactant in the elution buffer can be in the range of from about 2 mM up to about 200 mM, suitably from 10 mM up to 100 mM. The concentration of surfactant is preferably in the range of from 15 mM up to 50 mM.
The pH of the elution buffer is suitably in the range of from about 5 up to about 9, and preferably in the range of from 6 up to 8.
The total ionic strength of the elution buffer can be in the range of from about 0.1 up to 20 mS/cm, suitably from 1 up to 8 mS/cm, and preferably from 2 up to 5mS/cm.
The equilibration buffer, pretreatment buffer, sample washing buffer and the elution buffer can be the same or different. The concentration of surfactant, pH and total ionic strength can be the same or different than the values given for the elution buffer.
The process can be continuos, e.g. performed on a column, or batch-wise.
It is advantageous when carrying out the present invention to make use of a high temperature, since a high temperature more readily uncovers the strong complex between endotoxins and ApoA. The temperature is, however, limited to the range where irreversable denaturation of the protein does not occur. Thus, the temperature when carrying out the present invention, can be in the range of from about 2 up to about 95xc2x0 C., suitably from 15 up to 90xc2x0 C., preferably from 30 up to 85xc2x0 C., more preferably from 40 up to 75xc2x0 C.